1. Field of Invention
The invention relates to generation of micro-fluid droplets, e.g., for genetic analysis.
2. Description of Related Art
Sanger sequencing has improved greatly over the past 15 years but more significant advances are possible. Sanger sequencing was dramatically advanced by the transition to capillary array electrophoresis technologies. The utility of this separation method was also enhanced by the introduction of Energy Transfer (aka Big Dye) labels for the sequencing primers and terminators to enhance fluorescence signal strengths and reduce cross talk. These improvements in the labeling and separation coupled with many improvements in the other aspects of Sanger sequencing, facilitated the sequencing of the human genome and evolved the sequencing process to a relatively low cost and stable paradigm. However, this cost and production process is not sufficiently cheap or efficient to enable the routine sequencing or resequencing of a mammalian genome, so efforts are underway to reduce this cost. The injection of extension fragments into the capillary by conventional methods is only 1-0.1% efficient, wasting nearly all of the fluorescently labeled product because of the injection geometry. The current cloning, PCR or RCA sample preparation methods produce many orders of magnitude more template than is necessary and this scale requires the use of large space-demanding robotic transfer systems. The cloning/template amplification part of Sanger sequencing has not improved fundamentally since the start of the genome project.
Blazej et al. recently developed a sequencing microprocessor that performs thermal cycling of template in a 200 nL reactor. (Microfabricated bioprocessor for integrated nanoliter-scale Sanger DNA sequencing. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 7240-7245), incorporated by reference herein. The sequencing extension products are cleaned up on a microfabricated chip by electrophoresing them through a gel carrying a covalently attached oligonucleotide capture probe that selectively binds only the extension fragments. Electrophoretic “washing” eliminates excess salts, nucleotides and fluorescent labels. The products are released by raising the temperature of the device to the sequencing temperature which released the products for a cross channel injection. Using this method high quality sequencing data was obtained from only 1 femtomole of template. The efficiency is improved by placing the capture gel “in-line” with the separation column. See U.S. patent application Ser. No. 11/978,224, filed Oct. 25, 2007 and titled Inline-Injection Microdevice And Microfabricated Integrated Dna Analysis System Using Same, incorporated by reference herein. In this case, all of the extended fragments are captured within the separation column; subsequent thermal release provides 100% injection efficiency. This device produces high quality Sanger separations from only 100 attomoles of template.
Conventional methods, however, of PCR amplification do not allow single cell amplification to produce a uniform amount of template. Current “shake and bake” methods of generating emulsion droplets containing reactants (e.g., via agitation) produce polydisperse droplets, having a wide range of sizes and containing widely varying amounts of target and reagent.